ES2592352A1 - Procedure for the prevention and/or biological control of wilt caused by ralstonia solanacearum, through the use of bacteriophages useful for this and compositions thereof (Machine-translation by Google Translate, not legally binding) - Google Patents
Procedure for the prevention and/or biological control of wilt caused by ralstonia solanacearum, through the use of bacteriophages useful for this and compositions thereof (Machine-translation by Google Translate, not legally binding) Download PDFInfo
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- ES2592352A1 ES2592352A1 ES201530730A ES201530730A ES2592352A1 ES 2592352 A1 ES2592352 A1 ES 2592352A1 ES 201530730 A ES201530730 A ES 201530730A ES 201530730 A ES201530730 A ES 201530730A ES 2592352 A1 ES2592352 A1 ES 2592352A1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/40—Viruses, e.g. bacteriophages
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01M—CATCHING, TRAPPING OR SCARING OF ANIMALS; APPARATUS FOR THE DESTRUCTION OF NOXIOUS ANIMALS OR NOXIOUS PLANTS
- A01M7/00—Special adaptations or arrangements of liquid-spraying apparatus for purposes covered by this subclass
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G25/00—Watering gardens, fields, sports grounds or the like
- A01G25/02—Watering arrangements located above the soil which make use of perforated pipe-lines or pipe-lines with dispensing fittings, e.g. for drip irrigation
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G25/00—Watering gardens, fields, sports grounds or the like
- A01G25/06—Watering arrangements making use of perforated pipe-lines located in the soil
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G27/00—Self-acting watering devices, e.g. for flower-pots
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
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- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10211—Podoviridae
- C12N2795/10221—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10211—Podoviridae
- C12N2795/10231—Uses of virus other than therapeutic or vaccine, e.g. disinfectant
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Abstract
Description
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En el caso concreto del agente fitopatógeno R. solanacearum, cuyos hábitats son las plantas huésped y el suelo, un agente biológico que sea suministrado por medio del agua debe tener características biológicas que le permitan sobrevivir en ese medio, que no es el habitual ni de la bacteria ni de sus bacteriófagos específicos. Si dicha supervivencia resulta ser prolongada y los bacteriófagos mantienen su actividad lítica sobre el huésped tras largos períodos en el agua, además se favorece su aplicabilidad en el campo, ya que pueden vehicularse directamente a través de ese medio natural y simple como es el agua, sin necesidad de encapsularlos o de añadir otros soportes físicos y/o biológicos para proteger su viabilidad hasta el encuentro con la célula diana. Esa alta supervivencia facilitaría también la preparación de su presentación comercial, que podría ser en medio acuoso sin requerir condiciones de refrigeración (o incluso temperaturas inferiores) para su mantenimiento. In the specific case of the phytopathogenic agent R. solanacearum, whose habitats are host plants and soil, a biological agent that is supplied through water must have biological characteristics that allow it to survive in that environment, which is not the usual nor of the bacterium or its specific bacteriophages. If this survival proves to be prolonged and the bacteriophages maintain their lithic activity on the host after long periods in the water, their applicability in the field is also favored, since they can be directly transported through that natural and simple means such as water, without the need to encapsulate them or add other physical and / or biological supports to protect their viability until the encounter with the target cell. This high survival would also facilitate the preparation of your commercial presentation, which could be in an aqueous medium without requiring refrigeration conditions (or even lower temperatures) for maintenance.
Sin embargo, debe tenerse en cuenta que los bacteriófagos son parásitos intracelulares obligados y, como tales, necesitan de la célula huésped para su perpetuación. Puesto que se vehiculan hacia esta célula por diferentes vías, según los tipos de bacteriófagos y tipos de células huésped, es esperable un tiempo de supervivencia en el medio ambiente que les permita contactar con la célula huésped. Es conocido que este tiempo puede ser variable entre distintos bacteriófagos, siendo necesario su estudio en cada caso particular. Así, por ejemplo, se observan variaciones apreciables en la supervivencia de bacteriófagos del mismo serotipo/genotipo (Brion et al., 2002) o, incluso, entre bacteriófagos de bacterias acuáticas patógenas de peces, cuyo hábitat natural es el agua (Pereira et al., 2011). En este último caso se ha llegado a considerar como buena una supervivencia de los bacteriófagos de tres meses en agua, aceptándose que los bacteriófagos que presentan mayor supervivencia en agua son buenos candidatos para el control de enfermedades bacterianas de peces en sistemas de cultivo acuáticos (Pereira et al., 2011). However, it should be borne in mind that bacteriophages are obligate intracellular parasites and, as such, need the host cell for perpetuation. Since they are transported to this cell by different routes, depending on the types of bacteriophages and types of host cells, a survival time in the environment that allows them to contact the host cell is expected. It is known that this time can be variable between different bacteriophages, being necessary to study in each particular case. Thus, for example, there are appreciable variations in the survival of bacteriophages of the same serotype / genotype (Brion et al., 2002) or even among bacteriophages of pathogenic aquatic fish bacteria, whose natural habitat is water (Pereira et al ., 2011). In the latter case, a three month bacteriophage survival in water has been considered as good, accepting that bacteriophages that have greater water survival are good candidates for the control of bacterial diseases of fish in aquatic culture systems (Pereira et al., 2011).
Debe hacerse notar que R. solanacearum es una bacteria fitopatógena cuyo medio natural es el xilema de las plantas y con frecuencia el suelo, pero no el agua. Al no tratarse de una bacteria autóctona de ambientes acuáticos, no es esperable una alta supervivencia en agua de bacteriófagos específicos de la misma. De hecho, en ninguna de las publicaciones y patentes anteriormente comentadas se describe la viabilidad y actividad lítica de bacteriófagos líticos específicos de la especie anteriormente denominada R. solanacearum en agua medioambiental en ausencia de células huésped. It should be noted that R. solanacearum is a phytopathogenic bacterium whose natural environment is the xylem of plants and often the soil, but not water. Since it is not a native bacterium of aquatic environments, it is not expected a high survival in water of specific bacteriophages. In fact, none of the publications and patents mentioned above describe the viability and lithic activity of specific lytic bacteriophages of the species previously named R. solanacearum in environmental water in the absence of host cells.
Y, sin embargo, sería interesante disponer de bacteriófagos líticos específicos frente a R. solanacearum, particularmente que mostraran un amplio espectro de cepas de dicha And, nevertheless, it would be interesting to have specific lytic bacteriophages against R. solanacearum, particularly showing a broad spectrum of strains of said
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Por tanto, los tres bacteriófagos por separado, así como las combinaciones de los mismos, cumplen las características deseables para agentes de control biológico como son la alta especificidad por la célula huésped, no suponiendo un riesgo para la microbiota del agua, suelo o plantas, por ser específicos frente a R. solanacearum. Tampoco suponen una amenaza para la salud del ser humano, animales o plantas, al tratarse de virus bacteriófagos que sólo infectan bacterias. Son activos, además, en un rango de pH compatible con las características de distintos cursos fluviales de la geografía española, y en un rango de temperaturas compatible con las características de los mismos. Ello apoya el uso de los bacteriófagos de la presente invención, de forma individual o como combinaciones de los mismos, y el de las composiciones que comprenden dichos bacteriófagos, para el control de R. solanacearum, ya sea en agua de cursos naturales de agua como los ríos, arroyos o riachuelos, reservorios naturales de agua como lagos, lagunas, charcas, manantiales y acumulaciones subterráneas, reservorios artificiales de agua como embalses, depósitos cubiertos, tanques, estanques (con cubierta superficial o sin ella), pozos, agua de riego en general y reservorios de la misma como los reservorios naturales o artificiales antes citados. Therefore, the three bacteriophages separately, as well as the combinations thereof, meet the desirable characteristics for biological control agents such as high specificity for the host cell, not posing a risk to the microbiota of water, soil or plants, for being specific against R. solanacearum. Nor do they pose a threat to the health of humans, animals or plants, as they are bacteriophage viruses that only infect bacteria. They are also active in a pH range compatible with the characteristics of different river courses in the Spanish geography, and in a temperature range compatible with their characteristics. This supports the use of the bacteriophages of the present invention, individually or as combinations thereof, and that of the compositions comprising said bacteriophages, for the control of R. solanacearum, either in water of natural water courses such as rivers, streams or streams, natural water reservoirs such as lakes, lagoons, ponds, springs and underground accumulations, artificial water reservoirs such as reservoirs, covered deposits, tanks, ponds (with or without surface cover), wells, irrigation water in general and reservoirs of the same as the natural or artificial reservoirs mentioned above.
En ese sentido, los datos de campo recopilados por los presentes inventores sobre las aguas naturales contaminadas con R. solanacearum en las distintas comunidades autónomas españolas revelan que en los meses estivales (que es cuando se detecta la bacteria en agua y se prohíbe su uso para riego) las temperaturas diurnas más altas de estas aguas oscilan entre 13ºC y 17ºC, disminuyendo por la noche. Así, por ejemplo, en Salamanca y Guadalajara las temperaturas de las aguas muestreadas varían entre 14ºC y 4ºC. Además, en los distintos países del centro y norte de Europa con aguas medioambientales contaminadas con R. solanacearum las temperaturas son más bajas en los meses de verano. Por tanto, el rango de actividad observado para los bacteriófagos de la presente invención es compatible con su utilización en cursos naturales de agua, en particular de España. También lo es su rango de pH de actuación. Sin embargo, no es sencillo añadir bacteriófagos a un curso fluvial de agua en cantidad suficiente para conseguir el control efectivo de microorganismos en los mismos, en especial en el lugar concreto donde esa agua pueda desear utilizarse para regar, pues los bacteriófagos quedarán muy diluidos y serán transportados con el curso de agua a lo largo del mismo por lo que, aunque su tiempo de supervivencia sea muy alto, no se favorece que los bacteriófagos contacten con la célula huésped en el tramo de curso de agua que pueda ser de interés, salvo que los bacteriófagos se añadan a cursos de agua cortos y/o de reducido caudal, como riachuelos, regatos y canalizaciones artificiales, especialmente las que conduzcan a un reservorio donde el agua vaya a tener un tiempo In that sense, the field data collected by the present inventors on the natural waters contaminated with R. solanacearum in the different Spanish autonomous communities reveal that in the summer months (which is when the bacteria is detected in water and its use is prohibited for irrigation) the highest daytime temperatures of these waters range between 13ºC and 17ºC, decreasing at night. Thus, for example, in Salamanca and Guadalajara the temperatures of the sampled waters vary between 14ºC and 4ºC. In addition, in the different countries of central and northern Europe with environmental waters contaminated with R. solanacearum temperatures are lower in the summer months. Therefore, the range of activity observed for the bacteriophages of the present invention is compatible with its use in natural water courses, in particular in Spain. So is its range of acting pH. However, it is not easy to add bacteriophages to a watercourse in sufficient quantity to achieve effective control of microorganisms in them, especially in the specific place where that water may wish to be used to irrigate, since bacteriophages will be very diluted and they will be transported with the water course along it, so that although their survival time is very high, it is not favored that the bacteriophages contact the host cell in the watercourse section that may be of interest, except that bacteriophages be added to short and / or low flow water courses, such as streams, streams and artificial pipes, especially those that lead to a reservoir where the water will have time
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temperaturas de ensayo durante más de 5 meses, y que su supervivencia con actividad lítica alcance períodos de tres años. test temperatures for more than 5 months, and that their survival with lithic activity reaches periods of three years.
Por tanto, los bacteriófagos de la presente invención presentan como característica particularmente novedosa su supervivencia durante más de 5 meses en agua natural en ausencia de la célula huésped. Esto supone una característica adecuada y muy ventajosa para un agente de control biológico, que debe tener características que le permitan sobrevivir en el medio en el que se pretende aplicar que, en este caso, es el agua. Therefore, the bacteriophages of the present invention have a particularly novel feature as their survival for more than 5 months in natural water in the absence of the host cell. This is an appropriate and very advantageous feature for a biological control agent, which must have characteristics that allow it to survive in the environment in which it is intended to apply, which in this case is water.
Gracias a ello, es compatible con la aplicación del método y el uso de la presente invención que la composición que contiene los bacteriófagos se mantenga durante su almacenamiento y/o utilización, preferiblemente, a una temperatura comprendida en el rango de 4ºC y 24ºC, ambas incluidas, que puede considerarse un intervalo medioambiental habitual, aunque, dado que los bacteriófagos de la invención son activos hasta 31ºC, dicho intervalo puede extenderse al intervalo de 4ºC a 30ºC, ambos incluidos, a pesar de que este último valor no sea habitual en reservorios de agua medioambiental. Como ya se discutió anteriormente, también se consideran adecuadas condiciones donde la temperatura media del agua del reservorio esté comprendida entre 4ºC y 24ºC, ambos valores incluidos, dadas las fluctuaciones de temperatura ambiental diarias y estacionales. Esto facilita que las composiciones de la presente invención puedan mantenerse fácilmente durante largo tiempo previamente a su aplicación, en forma de suspensiones en las que los bacteriófagos se encuentran en un vehículo acuoso que puede ser agua (medioambiental, natural, destilada, previamente esterilizada, Thanks to this, it is compatible with the application of the method and the use of the present invention that the composition containing the bacteriophages be maintained during storage and / or use, preferably, at a temperature in the range of 4 ° C and 24 ° C, both included, which can be considered a usual environmental range, although, since the bacteriophages of the invention are active up to 31 ° C, said range can be extended to the range of 4 ° C to 30 ° C, both included, although the latter value is not usual in reservoirs of environmental water. As discussed earlier, conditions where the average water temperature of the reservoir is between 4ºC and 24ºC, both values included, are also considered, given the daily and seasonal ambient temperature fluctuations. This facilitates that the compositions of the present invention can be easily maintained for a long time prior to application, in the form of suspensions in which the bacteriophages are in an aqueous vehicle that can be water (environmental, natural, distilled, previously sterilized,
o sometida a otro tratamiento habitual para los vehículos acuosos) o una solución acuosa (tal como solución salina estéril, tampón fosfato salino, etc) y lista para utilizar y aplicar directamente cuando sea necesario. Por tanto, las composiciones de la presente invención, que pueden comprender cualquier vehículo o excipiente agronómicamente aceptable, pueden estar en forma líquida, por ejemplo en forma de suspensión acuosa, que se puede preparar en agua o en una solución acuosa y/o sus diluciones. Y en esta misma forma pueden usarse para el control de R. solanacearum y aplicarse con el método de prevención o tratamiento de la marchitez provocada por dicha bacteria, pudiendo estar por tanto listas para su aplicación directa desde su forma de comercialización y almacenamiento. or subjected to another usual treatment for aqueous vehicles) or an aqueous solution (such as sterile saline, phosphate buffered saline, etc.) and ready to use and apply directly when necessary. Therefore, the compositions of the present invention, which may comprise any agronomically acceptable carrier or excipient, may be in liquid form, for example in the form of an aqueous suspension, which can be prepared in water or in an aqueous solution and / or its dilutions . And in this same way they can be used for the control of R. solanacearum and applied with the method of prevention or treatment of the wilt caused by said bacterium, being able therefore to be ready for its direct application from its commercialization and storage form.
La elevada supervivencia, con mantenimiento de la actividad lítica sobre el huésped, de los bacteriófagos de la presente invención, favorece su aplicabilidad en el campo, ya que pueden vehicularse directamente a través del agua, un medio natural y simple, sin necesidad de encapsularlos o de añadir otros soportes físicos, químicos y/o biológicos The high survival, with maintenance of the lithic activity on the host, of the bacteriophages of the present invention, favors their applicability in the field, since they can be directly transported through water, a natural and simple means, without the need to encapsulate them or to add other physical, chemical and / or biological supports
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- CÓDIGO CEPA CEPA CODE
- PAÍS DE ORIGEN HUÉSPED AÑO COUNTRY OF ORIGIN GUEST YEAR
- Cepas internacionales International strains
- NCPPBa 1115 NCPPBa 1115
- Reino Unido (Ex Egipto) Patata 1961 United Kingdom (Ex Egypt) Potato 1961
- NCPPB 1584 NCPPB 1584
- Chipre Patata 1963 Cyprus Potato 1963
- NCPPB 2505 NCPPB 2505
- Suecia Patata 1972 Sweden Potato 1972
- NCPPB 2797 NCPPB 2797
- Suecia Solanum dulcamara 1974 Sweden Solanum dulcamara 1974
- BR 264 BR 264
- Reino Unido Solanum dulcamara 1993 United Kingdom Solanum dulcamara 1993
- Bordeaux 11-47 Bordeaux 11-47
- Francia Berenjena 1994 France Eggplant 1994
- Nantes 9-46 Nantes 9-46
- Francia Tomate 1994 France Tomato 1994
- 550 550
- Bélgica (Ex Turquía) Patata 1995 Belgium (Ex Turkey) Potato nineteen ninety five
- IPO-1609 IPO-1609
- Holanda Patata 1995 Holland Potato nineteen ninety five
- Port 448 Port 448
- Portugal Patata 1995 Portugal Potato nineteen ninety five
- W 12 W 12
- Bélgica Patata 1996 Belgium Potato nineteen ninety six
- WE 4-96 WE 4-96
- Reino Unido Agua de río 1996 United Kingdom River water nineteen ninety six
- Tom 1 Tom 1
- Reino Unido Tomate 1997 United Kingdom Tomato 1997
- Cepas españolas Spanish strains
- IVIAb-1602.1 IVIAb-1602.1
- Canarias Patata 1996 Canary Islands Potato nineteen ninety six
- IVIA-2049.53 IVIA-2049.53
- Canarias Suelo 1999 Canary Islands Ground 1999
- IVIA-2068.58a IVIA-2068.58a
- Canarias Patata 1999 Canary Islands Potato 1999
- IVIA-2068.61a IVIA-2068.61a
- Canarias Patata 1999 Canary Islands Potato 1999
- IVIA-2093.3.1 IVIA-2093.3.1
- Canarias Patata 1999 Canary Islands Potato 1999
- IVIA-2093.5T.1a IVIA-2093.5T.1a
- Canarias Patata 1999 Canary Islands Potato 1999
- IVIA-2128.1b IVIA-2128.1b
- Castilla-León Patata 1999 Castilla-Leon Potato 1999
- IVIA-2128.3a IVIA-2128.3a
- Castilla-León Patata 1999 Castilla-Leon Potato 1999
- IVIA-2167.1a IVIA-2167.1a
- Castilla-León Agua de río 1999 Castilla-Leon River water 1999
- IVIA-2167.2b IVIA-2167.2b
- Castilla-León Agua de río 1999 Castilla-Leon River water 1999
- IVIA-2528.A1-2 IVIA-2528.A1-2
- Castilla-León Agua de río 2001 Castilla-Leon River water 2001
- IVIA-2528.A3.1 IVIA-2528.A3.1
- Castilla-León Agua de río 2001 Castilla-Leon River water 2001
- IVIA-2528.54.A2 IVIA-2528.54.A2
- Castilla-León Agua de río 2001 Castilla-Leon River water 2001
- IVIA-2751.11 IVIA-2751.11
- Extremadura Agua de río 2003 Estremadura River water 2003
- IVIA-2762.1 IVIA-2762.1
- Extremadura Tomate 2003 Estremadura Tomato 2003
- IVIA-2762.4 IVIA-2762.4
- Extremadura Tomate 2003 Estremadura Tomato 2003
- IVIA-3090.1 IVIA-3090.1
- Andalucía Tomate 2005 Andalusia Tomato 2005
- IVIA-3090.5 IVIA-3090.5
- Andalucía Tomate 2005 Andalusia Tomato 2005
- IVIA-3205.A.22 IVIA-3205.A.22
- Castilla-La Mancha Agua de río 2006 Castilla la Mancha River water 2006
- IVIA-3243 IVIA-3243
- Andalucía Tomate 2006 Andalusia Tomato 2006
- IVIA-3359.9 IVIA-3359.9
- Castilla-La Mancha Agua de río 2007 Castilla la Mancha River water 2007
- IVIA-3359.10 IVIA-3359.10
- Castilla-La Mancha Agua de río 2007 Castilla la Mancha River water 2007
aNCPPB: National Collection of Plant Pathogenic Bacteria, Reino Unido. bIVIA: Colección de Bacterias del Instituto Valenciano de Investigaciones Agrarias, España. aNCPPB: National Collection of Plant Pathogenic Bacteria, United Kingdom. bIVIA: Collection of Bacteria of the Valencian Institute of Agricultural Research, Spain.
Las cepas de la NCPPB están disponibles en esta colección internacional. El resto de cepas está disponible en la colección de bacterias fitopatógenas del IVIA. NCPPB strains are available in this international collection. The rest of the strains are available in the collection of phytopathogenic bacteria of the IVIA.
También se ensayó la especificidad frente a otras especies de bacterias fitopatógenas y diversos aislados bacterianos de agua de río, para evaluar el posible impacto de los bacteriófagos aislados sobre la microbiota del agua natural. The specificity was also tested against other species of phytopathogenic bacteria and various bacterial isolates from river water, to assess the possible impact of isolated bacteriophages on the microbiota of natural water.
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VP4 y, sobre todo, Rhizobium RHEph01, todos ellos del tipo de los bacteriófagos similares a T7 (T7-like) (Tabla 4). Estas regiones (correspondientes al 5-23% del genoma completo de los bacteriófagos vRsoP-WF2, vRsoP-WM2 y vRsoP-WR2) pertenecen a regiones altamente conservadas. VP4 and, above all, Rhizobium RHEph01, all of them of the type of bacteriophages similar to T7 (T7-like) (Table 4). These regions (corresponding to 5-23% of the complete genome of the bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2) belong to highly conserved regions.
Tabla 4. Comparación de las secuencias de los genomas de los bacteriófagos vRsoP-WF2, vRsoP-WM2 y vRsoP-WR2 con genomas de varios bacteriófagos T7-like. Table 4. Comparison of the genome sequences of the bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 with genomes of several T7-like bacteriophages.
- Secuencia comparada (SEQ ID NO:) Sequence compared (SEQ ID NO :)
- Genoma del virus con la secuencia patrón de comparación (nº Acceso GenBank) Cobertura* Identidad** Genome of the virus with the standard sequence of comparison (No. GenBank Access) Coverage* Identity**
- 1 one
- Φ Ralstonia RSB1 (AB597179.1) 2% 84% Φ Ralstonia RSB1 (AB597179.1) 2% 84%
- 1 one
- Φ T7 (NC_001604.1) 5% 67% Φ T7 (NC_001604.1) 5% 67%
- 1 one
- Φ Rhizobium RHEph01 (JX483873.1) 19% 68% Φ Rhizobium RHEph01 (JX483873.1) 19% 68%
- 1 one
- Φ Vibrio VP4 (NC_007149.1) 5% 70% Φ Vibrio VP4 (NC_007149.1) 5% 70%
- 2 2
- Φ Ralstonia RSB1 (AB597179.1) 15% 66% Φ Ralstonia RSB1 (AB597179.1) fifteen% 66%
- 2 2
- Φ T7 (NC_001604.1) 5% 67% Φ T7 (NC_001604.1) 5% 67%
- 2 2
- Φ Rhizobium RHEph01 (JX483873.1) 23% 68% Φ Rhizobium RHEph01 (JX483873.1) 2. 3% 68%
- 2 2
- Φ Vibrio VP4 (NC_007149.1) 4% 70% Φ Vibrio VP4 (NC_007149.1) 4% 70%
- 3 3
- Φ Ralstonia RSB1 (AB597179.1) 15% 66% Φ Ralstonia RSB1 (AB597179.1) fifteen% 66%
- 3 3
- Φ T7 (NC_001604.1) 5% 67% Φ T7 (NC_001604.1) 5% 67%
- 3 3
- Φ Rhizobium RHEph01 (JX483873.1) 22% 68% Φ Rhizobium RHEph01 (JX483873.1) 22% 68%
- 3 3
- Φ Vibrio VP4 (NC_007149.1) 2% 70% Φ Vibrio VP4 (NC_007149.1) 2% 70%
* Homología entre las secuencias de los genomas comparados, en porcentaje. ** Nucleótidos coincidentes dentro de las zonas de homología de los genomas comparados, en porcentaje. * Homology between the sequences of the genomes compared, in percentage. ** Nucleotides matching within homology zones of the compared genomes, in percentage.
10 Estos resultados revelan que, excepto en estas zonas conservadas dentro de los bacteriófagos T7-like, los genomas de los bacteriófagos vRsoP-WF2, vRsoP-WM2 y vRsoP-WR2 contienen una secuencia nucleotídica altamente divergente de la de los demás bacteriófagos depositados en el GenBank. Por lo tanto, estas elevadas diferencias en secuencia nucleotídica avalan que los bacteriófagos vRsoP-WF2, vRsoP-WM2 y 10 These results reveal that, except in these conserved areas within T7-like bacteriophages, the genomes of the bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 contain a nucleotide sequence highly divergent from that of the other bacteriophages deposited in the Genbank. Therefore, these high differences in nucleotide sequence guarantee that the bacteriophages vRsoP-WF2, vRsoP-WM2 and
15 vRsoP-WR2 corresponden a una nueva especie dentro del género de los virus T7-like. 15 vRsoP-WR2 correspond to a new species within the genus of T7-like viruses.
36 36
con este patógeno, como en la prevención y/o control de la enfermedad que causa en campo. Esta capacidad de biocontrol es especialmente importante si se tiene en cuenta que no hay actualmente métodos de control eficaces en suelo, ni disponibles en agua. Y, en el presente caso, como se ha discutido previamente y se demuestra en los experimentos anteriores, los agentes de biocontrol proporcionados por la presente invención presentan la inesperada característica de su elevada supervivencia en agua en condiciones de temperatura medioambiental habituales en España, lo que es una ventaja tanto para su aplicación a las plantas mediante el agua de riego y para el control y prevención de la presencia de R. solanacearum en la misma, como para el fácil y prolongado mantenimiento de las formas de comercialización de los bacteriófagos de la invención previamente a su utilización. Dicho mantenimiento puede tener lugar en medio acuoso durante largo tiempo sin pérdidas severas de la actividad lítica y ni siquiera requeriría, antes de su aplicación directa al agua, de la dilución previa de los bacteriófagos o de su mezcla con algún tipo de soporte físico o químico que actuara como vehículo para facilitar su interacción con la bacteria diana o que garantizara su estabilidad hasta conseguirlo, de forma que la aplicación al agua de riego o a corrientes o reservorios de agua en los que se quiera controlar posibles contaminaciones con R. solanacearum podría ser, por ejemplo, por simple vertido sobre dicha agua. with this pathogen, as in the prevention and / or control of the disease it causes in the field. This biocontrol capacity is especially important if one considers that there are currently no effective control methods in soil, nor available in water. And, in the present case, as previously discussed and demonstrated in the previous experiments, the biocontrol agents provided by the present invention have the unexpected characteristic of their high survival in water under usual environmental temperature conditions in Spain, which It is an advantage both for its application to the plants through irrigation water and for the control and prevention of the presence of R. solanacearum in it, as well as for the easy and prolonged maintenance of the commercialization forms of the bacteriophages of the invention prior to use. Such maintenance can take place in aqueous medium for a long time without severe losses of lithic activity and would not even require, prior to its direct application to water, the prior dilution of bacteriophages or their mixing with some type of physical or chemical support. that acts as a vehicle to facilitate its interaction with the target bacterium or that guarantees its stability until it is achieved, so that the application to irrigation water or water streams or reservoirs in which it is possible to control possible contamination with R. solanacearum could be, for example, by simple pouring on said water.
Depósito de microorganismos Deposit of microorganisms
Los bacteriófagos vRsoP-WF2, vRsoP-WM2 y vRsoP-WR2, con capacidad para lisar células de R. solanacearum han sido depositados en la colección alemana de cultivos microbianos Leibniz-Institut DSMZ-Deutsche-Sammlung von Mikro-organismen und Zellkulturen GmbH, Inhoffenstrasse 7B, 38124 Braunschweig, Alemania, siguiendo las normas del Tratado de Budapest para el depósito de microorganismos para fines de patentes, en las siguientes fechas y se les ha asignado el siguiente número de acceso (Tabla 5). The bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2, with the ability to lyse R. solanacearum cells have been deposited in the German microbial culture collection Leibniz-Institut DSMZ-Deutsche-Sammlung von Mikro-organism und Zellkulturen GmbH, Inhoffenstrasse 7B, 38124 Braunschweig, Germany, following the rules of the Budapest Treaty for the deposit of microorganisms for patent purposes, on the following dates and have been assigned the following access number (Table 5).
Tabla 5. Datos del depósito de los bacteriófagos en la colección alemana DSMZ. Table 5. Bacteriophage deposit data in the German DSMZ collection.
- Material Material
- Fecha de Depósito Número de acceso Deposit date Access number
- vRsoP-WF2 vRsoP-WF2
- 15 de abril de 2015 DSM 32039 April 15, 2015 DSM 32039
- vRsoP-WM2 vRsoP-WM2
- 15 de abril de 2015 DSM 32040 April 15, 2015 DSM 32040
- vRsoP-WR2 vRsoP-WR2
- 15 de abril de 2015 DSM 32041 April 15, 2015 DSM 32041
40 40
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| US15/576,798 US10508266B2 (en) | 2015-05-26 | 2016-05-24 | Method for the prevention and/or the biological control of bacterial wilt caused by Ralstonia solanacearum, via the use of bacteriophages suitable for this purpose and compositions thereof |
| PCT/ES2016/070392 WO2016189180A1 (en) | 2015-05-26 | 2016-05-24 | Method for the prevention and/or the biological control of wilt caused by ralstonia solanacearum, via the use of bacteriophages suitable for this purpose and compositions thereof |
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| CN108410825B (en) * | 2018-04-20 | 2022-09-20 | 南京农业大学 | Phage cocktail and application thereof |
| CN112029789B (en) * | 2020-08-03 | 2022-05-31 | 华南农业大学 | Application of phage trp574 gene in reducing resistance of ralstonia solanacearum to phage |
| CN115261338B (en) * | 2022-08-15 | 2023-09-22 | 福建省农业科学院植物保护研究所 | Lytic phage S5 with tobacco bacterial wilt prevention and control function and application thereof |
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| JP2007252351A (en) * | 2006-02-24 | 2007-10-04 | Hiroshima Univ | Ralstonia solanacearum infective bacteriophage |
| KR20120055801A (en) * | 2010-11-24 | 2012-06-01 | 동아대학교 산학협력단 | Bacteriophage having specific lysis activity for bacterial wilt pathogen in plant |
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| JP2007252351A (en) * | 2006-02-24 | 2007-10-04 | Hiroshima Univ | Ralstonia solanacearum infective bacteriophage |
| KR20120055801A (en) * | 2010-11-24 | 2012-06-01 | 동아대학교 산학협력단 | Bacteriophage having specific lysis activity for bacterial wilt pathogen in plant |
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| BAE JU YOUNG et al. Biocontrol Potential of a Lytic Bacteriophage PE204 against Bacterial Wilt of Tomato.Journal of Microbiology and Biotechnology DEC 2012 00/12/2012 VOL: 22 No: 12 Pags: 1613-1620 ISSN 1017-7825(print) ISSN 1738-8872(electronic) Doi: doi:10.4014/jmb.1208.08072 * |
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| CN110564875B (en) * | 2019-08-09 | 2023-08-18 | 广西壮族自治区农业科学院 | Primer group, reagent, kit and application capable of rapidly detecting bacterial wilt diseases of mulberries |
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